The overall goal of this research is to develop a mechanistic understanding of Golgi membrane protein retention, the role of ion gradients in membrane traffic, and the assembly, transport and targeting of the VATPase in the simple eukaryote Saccharomyces cerevisiae. Yeast has proved to be an excellent model system, both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. Genetic analysis has revealed a large collection of yeast genes encoding not only subunits of the yeast vacuolar proton-translocating ATPase (V-ATPase), but also four (4) genes encoding proteins that are not subunits of the enzyme but instead are localized to the endoplasmic reticulum (ER) and required for assembly and transport of the V-ATPase. Using both genetic and biochemical approaches, the V-ATPase assembly factors will be characterized to determine which factors function in specific steps of the assembly process. These assembly factors will also be characterized for a role in the loading of the V-ATPase into ER-derived vesicles, and in escorting the V-ATPase to the Golgi complex. There are two different forms of the yeast V-ATPase; the Golgi and endosomal form of the complex assembles with the Stv1p isoform of the 100 kDa subunit, and the complex on the vacuole membrane assembles with the Vph1p isoform of the 100 kDa subunit. The Golgi/endosomal localization signals in the Stv1p isoform will be identified by mutational analysis, and the proteins that recognize and sort the Stv1-associated V-ATPase will be identified by genetics and characterized. pH-sensitive forms of the Green Fluorescent Protein (GFP) will be targeted to the various yeast intracellular organelles to determine the pH of the Golgi-endosomal network. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of membrane transport in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. These basic studies in yeast are providing important insights into our understanding of many diseases in humans related to defects in organelle acidification.